Epidemic keratoconjunctivitis is caused by human adenovirus species D (HAdV-D) types 8, 19, and 37, and more recently also 53 and 54. The clinical manifestations of this infection include severe membranous conjunctivitis and epithelial keratitis, followed by multifocal subepithelial (stromal) corneal infiltrates that cause photophobia and reduced vision and may persist for months to years. Previous studies focused on cellular and nuclear signaling events that led to expression of proinflammatory mediators such as CXCL1 (mouse), CXCL8 (human), and CCL2 in the corneal stroma. However, recent work by our laboratory using the mouse adenovirus keratitis model we developed and characterized in the last funding cycle resulted in novel observations regarding specific cytokine expression signatures associated with different pathogen associated molecular patterns presented to resident corneal stromal cells. Based on these observations, we propose that stromal inflammation following corneal adenovirus infection represents the summation of specific cytokine responses to infection by phenotypically distinct cell types in the corneal stroma. Our proposed study of the specific mechanisms for response of corneal stromal bone marrow derived cells to distinct adenovirus associated molecular patterns will be unique. In addition, we will employ for these studies a highly novel three dimensional model of tissue inflammation, the human corneal facsimile. Our specific aims to accomplish our goal are to test the following hypotheses: 1) adenoviruses infect bone marrow derived cells in the corneal stroma, 2) adenoviruses induce CXCL-1/8 chemokine expression in the corneal stroma through a molecular interaction between viral capsid protein and keratocytes and 3) adenoviruses induce CCL2 and IL-6 expression in the corneal stroma through a molecular interaction between viral DNA and dendritic cells. These studies will utilize both the mouse model of adenovirus keratitis and the human corneal facsimile model, and will uniquely test cellular and molecular mechanisms of innate immunity in a disease model of adenovirus infection. As adenoviruses represent the most common cause of eye infections, this proposal will address a major public health concern. PUBLIC HEALTH RELEVANCE: Ocular surface infection by adenoviruses represents the most common type of eye infection, and affected patients suffer considerably from pain and blurred vision when the cornea is involved. However, there is at present no specific therapy against adenovirus infections or the ocular inflammation that follows. Corticosteroids represent the only available treatment for adenovirus keratitis, but can be associated with unwanted effects, including prolonged adenovirus replication. This proposal directly addresses the mechanism of corneal innate immune responses to adenovirus infection of the cornea, specifically the interactions between specific pathogen associated molecular patterns on the adenovirus and resident bone marrow derived cells in the corneal stroma. This work will inform a mechanistic molecular approach to future translational therapies for adenovirus induced corneal inflammation.